Non-infectious, protease defective HIV particles and nucleic acid molecules encoding therefor

ABSTRACT

This invention is directed toward mutated DNA, proteins, or protein fragments and particles from the L-2 cell line. The invention is also directed to diagnostic, prophylactic and therapeutic methods of making and using the DNA, proteins and particles.

[0001] This application is based on, and claims the benefit of, U.S.Provisional Application No. 60/043,047, filed Apr. 4, 1997, and entitledNON-INFECTIOUS, PROTEASE DEFECTIVE HIV PARTICLES AND NUCLEIC ACIDMOLECULES ENCODING THEREFOR.

BACKGROUND OF THE INVENTION

[0002] This invention relates to human immunodeficiency viruses ingeneral and, more specifically to non-infectious vaccines for thetreatment of human immunodeficiency disease.

[0003] AIDS is a lethal disease caused by human immunodeficiency virusesHIV-1 and HIV-2. This disease is characterized by a long, latentasymptomatic phase. During this phase the HIV particles are suppressedby the body's immune defense system. The next phase of the diseaseoccurs when the body's immune system is no longer able to suppress theviral particles. At this stage of the disease, the HIV viral particlesattack and destroy a key component of the body's immune defense systemcalled CD4⁻ cells. Once these cells are destroyed, the third and finalphase of the disease occurs. During this last phase, the body isextremely susceptible to infections by many different kinds of diseases.These so-called secondary infections are the cause of death in many AIDSpatients.

[0004] One of the central mysteries about AIDS is why HIV particles canexist in the body for so long, yet the patient remains asymptomatic. Asa corollary to this riddle, there is no certain explanation why theimmune system suddenly fails to be effective against the particles, thusenabling the second phase of the disease.

[0005] One theory for the sudden onslaught of the HIV particles on theCD4⁺ cells is that this onslaught is made by “defective” HIV particles.These new, defective particles are not recognized by the body's immunesystem. These defective particles have mutations in some of the keypolypeptides of an HIV viral particle, yet somehow are still able toaffect sufficient damage on the CD4⁺ cells by means other than theclassical invasion of the cells.

[0006] Surprisingly, non-infectious HIV particles produced by a cellcalled “L-2” fuse more efficiently than wild-type HIV with asubpopulation of CD4⁺ cells.

[0007] The enhanced fusion is caused by the 4-fold increased level ofgp120, the two deletion mutations in gp120, altered conformationalepitopes of gp120 and/or a combination of these factors, as well asmutant forms of Nef, gp41, protease, and Vpr, as discussed below. Thefusion is far from the typical action of normal HIV infectious viralparticles, in that it leads to the stimulation and release of II-2R, Fasligand and interferon-gamma 1 from a small subset of CD4⁺ cells (Kameokaet al., International Immunology 9(10):1453-1462 (1997)). This occursafter L-2 particle fusion with these CD4⁺ cells, a high percentage ofuninfected, healthy neighboring CD4⁺, as well as CD8⁺ cells, areprematurely thrown into a programmed cell death cycle, ultimatelyresulting in apoptosis. Apoptosis is a natural process in most cells inthe body, but only after such cells have become senescent. Fusion of L-2particles with CD4⁺ cells can also cause such cells to formmultinucleated protoplasmic masses called “syncytia”, which die shortlythereafter.

[0008] Thus, there exists a need to detect such defective HIV particlesin the infected population; both for asymptomatic carriers, as well asfor ARC (AIDS elated Complex) or AIDS patients. Furthermore, a needexists for prophylactic, immunogenic, and therapeutic agents for suchdefective particles, as well as polypeptides or fragments ofpolypeptides derived from the particles or their mutant nucleic acidsequences. This need exists due to the fact that mutations found in L-2particles are normally produced in HIV infected individuals, so thatdevelopment of immunity to them by natural or artificial means is a keyto thwarting the pathogenic process leading to ARC and AIDS. Thisinvention provides these advantages and more.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 sets forth the mutations in the nucleotide sequences anddeduced amino acid residues of the defective HIV-1 provirus in L-2 cellscompared with those in MO/LAI and LAI cells. The portions of thenucleotide sequences of the relevant pol protease (prot.), vpr, env(gp120 and gp41), and nef gene regions in wild-type HIV-1 (LAI), as wellas the proviruses of L-2 or MO/LAI, are shown in the upper part for eachgene. The portions of the deduced amino acid sequences of Pol, Vpr, Envand Nef polypeptides setting forth the mutations are shown below thenucleotide sequences. The wild-type LAI control sequences are taken frompreviously reported sequences for HIV-1 (LAI), in GenBank. The sequencesidentical with those of wild-type LAI are indicated by dots. An asterisk(*) indicates the appearance of stop codons. For the upper part, a dash(−) indicates a deletion in nucleotide sequences. For the amino acidsequences represented in the lower part, a dash indicates sequencesidentical with the wild type HIV-1 (LAI) sequence. A hatchmark (#)indicates a deletion in deduced amino acid residues. Three pluses (+)indicates any three nucleotides coding for the corresponding amino acidshown in the deduced amino acid sequence.

[0010]FIG. 2 sets forth the primers used for PCR amplification ofprotease-defective HIV-1 provirus in L-2 cells. Nucleotide numbers arethose of the HIV-1 molecular clone pNL432 (Adachl et al., J. Virol,59:284-291 (1986)).

[0011]FIG. 3 sets forth the partial nucleic acid sequence of the mutatednef gene of the HIV-1 protease-defective provirus in the L-2 cell, wherethe individual bases that differ from the wild-type LAI HIV-1 virus inGenBank are encircled.

[0012]FIG. 4 sets forth the sequence of the truncated Nef polypeptidecoded for by the mutated nef gene of the L-2 HIV-1 provirus. Theencircled amino acids differ from the wild-type LAI HIV-1 virus aminoacid sequence set forth in GenBank.

SUMMARY OF THE INVENTION

[0013] This invention is directed to nucleic acid molecules andfragments thereof containing mutations in certain genes of the HIV-1virus, and the polypeptides and fragments thereof that are encoded bysuch mutated nucleic acid molecules, antibodies to such polypeptides,nucleic acid and antibody-based assays, as set forth below, as well astherapeutic uses of such antibodies. This invention is also directed toimmunogens, and compositions of immunogens and adjuvants, containingsuch mutated HIV-1 viruses and in the production and use in therapeuticand prophylactic (immunogenic) methods and compositions.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention is directed to the identification of mutationswithin pathologically important genes of the human immunodeficiencygenome (HIV) which render viral particles non-infectious. Thenon-infectious phenotype of the viral particles can advantageously beused as a natural source of material for the production of immunogensfor vaccination against or treatment of Acquired ImmunodeficiencySyndrome (AIDS). The mutations identified within the variouspathologically important genes can also be advantageously used toengineer new non-infectious viral particles for use as immunogensagainst essentially any type of HIV isolate.

[0015] In one embodiment, the invention is directed to the mutationsdiscovered in various pathologically important genes of thenon-infectious, protease-defective HIV-1 provirus harbored by L-2 cells.The partial sequences illustrating the nucleotide and amino acid changescaused by these mutations are set forth in FIGS. 1, 3 and 4.

[0016] The formation of syncytia and apoptosis of bystander T cellsappear to be a mechanism of HIV-1-induced pathogenesis, leading to thediminution of T cells. (Amazon and Capon, Immunol. Today, 12:102-105(1991); Malderelli et al., J. Virology, 69:6457-6465 (1995); andSodroski et al., Nature, 322:470-474 (1996). It has been reported thatsubclones obtained from surviving cells after MT-4, a CD4 human T cellline, had been infected with wild-type LAI or infectious molecular clonepNLA32-derived viruses, were predominantly producers of infectious butless cytopathogenic virus particles (Yunoki et al., Arch. Virol.,116:143-158 (1991); Nishino et al., Arch. Virol., 120:181-192 (1991)).The provirus in these persistently infected cell clones carriedmutations in accessory genes, such as vif, vpr, and vpu. (Nishino etal., J. Gen. Virol., 75:2241-2251 (1994), and Kishi et al., J. Virol.,69:7507-7518 (1995)). However, about 10% of the subclones produced abovewith LAI virus showed defective phenotypes even in essential geneproducts. In particular, one of these, named the L-2 cell clone, was alarge-scale producer of protease-defective, gp120-containingnoninfectious particles. (Ikuta et al., J. Cancer Res., 78:118-123(1988)).

[0017] Surprisingly, the protease-defective viral particles producedfrom the L-2 cell clone rapidly induce syncytium formation of uninfectedT cells by virus-to-cell fusion (fusion from without), without the needfor viral replication. (Ohki et al., J. AIDS, 4:1233-1240 (1991). Inaddition, the L-2 particles have a significantly higher (>fivefold)activity for apoptosis induction in primary peripheral blood T cells, aswell as a certain subset of U937 cells when compared with solublerecombinant gp120 or the wild-type HIV-1 (LAI) strain (Kameoka et al.,Int. Immunol., 11:1687-1697 (1996); Kameoka et al., J. Clin. Microbiol.,35:41-47 (1997)). Sodium dodecyl sulfate-polyacrylamide gelelectrophoretic (SDS-PAGE) analysis demonstrates that the Gag precursorpolypeptide in L-2 particles was not cleaved to mature Gag polypeptidesand that the Env/Gag ratio in L-2 particles was about fourfold higherthan that in HIV-1 (LAI) wild-type particles produced from persistentlyinfected MOLT-cells (Yunoki et al., Arch. Virol., 116:143-158 (1991);Kameoka et al. (1997) supra)).

[0018] “Substantially pure” when used to describe the state of theclaimed nucleic acids, polypeptides or fragments thereof, is intended tomean that the claimed molecules are free of at least a portion of thecontents associated with or occurring with the claimed nucleic acids,polypeptides or fragments thereof in the native environment.

[0019] As used herein, the term “substantially” or “substantially thesame” when used in reference to a nucleotide or amino acid sequence isintended to mean that the nucleotide or amino acid sequence shows aconsiderable degree, amount or extent of sequence identity when comparedto a reference sequence. Such considerable degree, amount or extent ofsequence identity is further considered to be significant and meaningfuland therefore exhibit characteristics which are definitivelyrecognizable or known. Thus, a nucleotide sequence which issubstantially the same nucleotide or amino acid sequence as a claimedsequence, refers to a sequence which exhibits characteristics that aredefinitively known or recognizable as representing the claimednucleotide or amino acid sequence and minor modifications thereof.

[0020] In regard to substantially the same nucleotide sequences, suchcharacteristics include for example, specific hybridization of anucleotide sequence to a claimed nucleic acid or its compliment.Hybridization principles and methods for determining hybridizationspecificity are well established and are known by one skilled in theart. Such principles and methods can be found in, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1992), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989). Thus, itis not necessary that two nucleic acids exhibit sequence identity to besubstantially complimentary, only that they can specifically hybridizeor be made to specifically hybridize without detectible cross reactivitywith other similar sequences.

[0021] In regard to substantially the same amino acid sequence, suchcharacteristics which are definitively recognizable or known include,for example, maintaining the biological function of the polypeptide orsubstitution of conservable amino acid residues. It is understood thatlimited modifications may be made without destroying the biologicalfunction of the claimed polypeptides or fragments thereof, and that onlya portion of the entire primary structure can be required in order toeffect activity. For example, the claimed polypeptides of the inventionhave an amino acid sequence substantially similar to those indicated forthe L-2 polypeptides shown in FIGS. 1 or 4, but minor modifications ofthese sequences which do not destroy their activity also fall within thedefinition of the polypeptides claimed as such. Moreover, fragments ofthe sequences of the L-2 polypeptides shown in FIGS. 1 and 4, aresimilarly included within the definition. It is understood that minormodifications of primary amino acid sequence may result in polypeptideswhich have substantially equivalent or enhanced function as compared tothe sequences of the L-2 polypeptides set forth in FIGS. 1 and 4. Thesemodifications can be deliberate, as through site-directed mutagenesis,or may be accidental such as through mutation in hosts which produce theclaimed polypeptides. All of these modifications are included as long asthe biological functions are retained.

[0022] As used herein, the term “fragment” when used in reference to anucleic acid encoding the claimed polypeptides is intended to mean anucleic acid having substantially the same sequence as a portion of anucleic acid encoding the claimed Pol protease, Nef, Env gp41, Env gp120or Vpr polypeptides or fragments thereof. The nucleic acid fragment issufficient in length and sequence to selectively hybridize to a Polprotease, Nef, Env gp41, gp120 or Vpr encoding nucleic acid or anucleotide sequence that is complimentary to a Pol protease, Nef, Envgp41, gp120 or Vpr encoding nucleic acid. Therefore, fragment isintended to include primers for sequencing and polymerase chain reaction(PCR) as well as probes for nucleic acid blot or solution hybridization.

[0023] As used herein, the term “functional fragment” when used inreference to a Pol protease, Nef, Env gp41, gp120 or Vpr polypeptide isintended to refer to a portion of a Pol protease, Nef, Env gp41, gp120or Vpr polypeptide which still retains some or all or the apoptoticinducing activity of the L2 particles containing one or more of thesepolypeptides as described herein. The term is also intended to includepolypeptides that include, for example, modified forms of naturallyoccurring amino acids such as D-steroisomers, non-naturally occurringamino acids, amino acid analogues and mimics so long as suchpolypeptides retain functional activity as defined above.

[0024] The HIV-1 provirus from L-2 cells has been analyzed with respectto genetic mutations that result in non-infectious HIV particles. Genesof HIV-1 provirus from L-2 cells have been isolated and sequenced andcompared to the wild type HIV-1 MO/LAI and LAI isolates. Variousmutations have been observed in several viral genes encoding viralpolypeptides, including the nef, env, pol and vpr genes.

[0025] One aspect of the instant invention is a substantially purenucleic acid molecule encoding a mutant HIV-1 Nef protein, containingthe encircled L-2 mutations set forth as SEQ ID NO: 45 (FIG. 3), and anyfragments of such a nucleic acid molecule containing at least one of theencircled L-2 mutations which can be used to specifically detect themutated nucleic acid or protein.

[0026] The nef gene of HIV-1 provirus from L-2 cells was compared to thewild type HIV-1 in MO/LAI and LAI isolates GenBank accession No. K02013.Several mutations in the nef gene compared to the wild type HIV-1 nefgene sequence were observed. One mutation is the substitution of A for Gat nucleotide 169 of the nef gene. This mutation results in a stop codonat amino acid residue 57 such that the Nef polypeptide is truncated andcontains amino acids 1 through 56.

[0027] The invention provides a substantially pure nucleic acid moleculeor fragment thereof that encodes a truncated Nef protein comprising anucleic acid sequence that encodes substantially the same sequence ofamino acid residues 1 through 56 of an HIV-1 Nef polypeptide.

[0028] Another mutation found in the nef gene of HIV-1 provirus from L-2cells is the substitution of G for the A at nucleotide 43 of the wildtype nef gene. This mutation changes Thr at amino acid residue 15 of theNef polypeptide to Ala. An additional mutation found in the nef gene ofHIV-1 provirus from L-2 cells is the substitution of A for the G atnucleotide 56 of the wild type nef gene. This mutation changes Arg atamino acid residue 19 of the Nef polypeptide to Lys.

[0029] Additional mutations of the nef gene of HIV-1 provirus from L-2cells were found. One of these mutations is the substitution of T forthe C at nucleotide 98 of the wild type nef gene. This mutation changesAla at amino acid residue 33 of the Nef polypeptide to Val. Stillanother of these mutations is the substitution of A for the C atnucleotide 161 of the wild type nef gene. This mutation changes Ala atamino acid residue 54 of the Nef polypeptide to Asp.

[0030] An additional mutation found in the nef gene of HIV-1 provirusfrom L-2 cells is the substitution of A for the G at nucleotide 111 ofthe wild type nef gene. This mutation results in no change in the aminoacid at residue 37 of the encoded Nef polypeptide since the nucleotidesubstitution occurs in the third position of the codon for Leu and allnucleotide substitutions at this position would encode for Leu.Therefore, nucleotide 111 of the nef gene can be any of the fournucleotides.

[0031] The invention also provides a substantially pure nucleic acid orfragment thereof that encodes a Nef polypeptide comprising a nucleotidesequence that encodes substantially the same amino acid sequence of anHIV-1 Nef polypeptide containing Ala at amino acid residue 15. Theinvention additionally provides a substantially pure nucleic acid orfragment thereof that encodes a Nef polypeptide comprising a nucleotidesequence that encodes substantially the same amino acid sequence of anHIV-1 Nef polypeptide containing Lys at amino acid residue 19.

[0032] The invention further provides a substantially pure nucleic acidor fragment thereof that encodes a Nef polypeptide comprising anucleotide sequence that encodes substantially the same amino acidsequence of an HIV-1 Nef polypeptide containing Val at amino acidresidue 33. The invention also provides a substantially pure nucleicacid or fragment thereof that encodes a Nef polypeptide comprising anucleotide sequence that encodes substantially the same amino acidsequence of an HIV-1 Nef polypeptide containing Asp at amino acidresidue 54.

[0033] The nucleotide substitutions and amino acid mutations found inthe nef gene of HIV-1 provirus from L-2 cells and the encoded Nefpolypeptide are shown in FIGS. 3 and 4, respectively.

[0034] A second aspect of the instant invention is a substantially purenucleic acid molecule encoding a mutated HIV-1 env gp41 proteincontaining the L-2 mutation set forth in SEQ ID NO: 29 (FIG. 1) and anyfragment of such nucleic acid molecule containing the mutation which canbe used to specifically detect the mutated nucleic acid or protein.

[0035] A mutation in the L-2 cell derived HIV-1 provirus env geneencoding the Env gp41 polypeptide has also been identified. The mutationis the substitution of G for the A at nucleotide 7781 (nucleotide 1979in FIG. 1) in a wild type env gene. This mutation changes Lys at aminoacid residue 660 of the Env gp41 polypeptide to Arg.

[0036] The invention also provides a substantially pure nucleic acid orfragment thereof that encodes an Env gp41 polypeptide comprising anucleotide sequence that encodes substantially the same amino acidsequence of an HIV-1 Env gp41 polypeptide containing Arg at amino acidresidue 660.

[0037] A third aspect of the instant invention is a substantially purenucleic acid molecule encoding a mutated HIV-1 pol protease proteincontaining the L-2 mutation set forth in SEQ ID NO:5 (FIG. 1) and anyfragment of such nucleic acid molecule containing the mutation which canbe used to specifically detect the mutated nucleic acid or protein.

[0038] A mutation in the L-2 cell derived HIV-1 provirus pol proteasegene encoding the pol protease polypeptide has also been identified. Themutation is the insertion of T after the A at nucleotide 41 in wild typepol protease gene. This mutation results in a frameshift and theappearance of a stop codon at amino acid residue 30 of the Pol proteasepolypeptide. This results in a 29 amino acid Pol protease polypeptidewith the carboxyl-terminal amino acid sequenceAsnAspArgGlyAlaThrLysGlySerSerIleArgTyrArgSerArg (SEQ ID NO:6) startingat amino acid residue 14.

[0039] The invention additionally provides a substantially pure nucleicacid or fragment thereof that encodes a Pol protease polypeptidecomprising a nucleotide sequence that encodes substantially the samesequence of amino acid residues 1 through 13 of an HIV-1 Pol proteasepolypeptide fused at the carboxyl-terminus to substantially the sameamino acid sequence AsnAspArgGlyAlaThrLysGlySerSerIleArgTyrArgSerArg(SEQ ID NO:6).

[0040] A fourth aspect of the invention is directed to a substantiallypure nucleic acid molecule encoding a mutant HIV-1 gp120 proteincontaining the L-2 mutations set forth in SEQ ID NOS:17 and 23 (FIG. 1),and any fragment of said nucleic acid containing one or more of the L-2mutations which can be used to specifically detect the mutated nucleicacid or protein.

[0041] Mutations in the L-2 cell derived HIV-1 provirus env geneencoding the Env gp120 O polypeptide have also been identified. A mutantEnv gp120 found in HIV-1 provirus from L-2 cells has a substitution ofArg for the Ser at amino acid residue 143, substitution of Ile for theMet at position 153 and deletion of eight amino acids, from 144 through151, of the wild type HIV-1 from LAI cells as well as substitution ofVal for the Ile at amino acid residue 187 and deletion of two aminoacids, 192 and 193, of the wild type HIV-1 from LAI cells. Thesubstitution of Arg at amino acid position 143 occurred due tonucleotide substitutions that changed the codon for Ser to the codon forArg. Any mutation that results in changing the nucleotides encoding Ser143 (nucleotides 427 through 429) to Arg can be used in the nucleicacids of the invention. For example, nucleotides 427 through 429, AGT,can be substituted with nucleotides that result in a codon for Arg, suchas substitution of A for the T at nucleotide 429, substitution of G forthe T at nucleotide 429, substitution of C for the A at nucleotide 427,substitution of C for the A at position 427 combined with substitutionof C for the T at nucleotide 429, substitution of C for the A atposition 427 combined with the substitution of A for the T at nucleotide429, and substitution of C for the A at position 427 combined with thesubstitution of G for the T at nucleotide 429, all of which result insubstitution of Arg for Ser at amino acid position 143. The substitutionof Ile for Met at amino acid residue 153 results from substitution of Afor the G at nucleotide 459. The substitution of Val for Ile at aminoacid residue 187 results from substitution of G for A at nucleotide 559.This mutant Env gp120 mutant is identical to a Env gp120 mutant found inHIV-1 from MO/LAI (see FIG. 1).

[0042] The invention also provides a substantially pure nucleic acid orfragment thereof that encodes an Env gp120 polypeptide comprising anucleotide sequence that encodes substantially the same amino acidsequence of an HIV-1 Env gp120 polypeptide containing Arg at amino acidresidue 143, Ile at amino acid residue 153, and Val at the amino acidresidue corresponding to amino acid residue 187 of a wild type HIV-1 Envgp120 polypeptide and absent amino acid residues corresponding to aminoacid residues 144 through 151 and 192 through 193 of a wild type HIV-1Env gp120 polypeptide.

[0043] A fifth aspect of the invention is directed to a substantiallypure nucleic acid molecule encoding a mutant HIV-1 Vpr proteincontaining the L-2 mutation set forth in SEQ ID NO: 11 (FIG. 1), and anyfragment of such a nucleic acid molecule containing the L-2 mutationwhich can be used to specifically detect the mutated nucleic acid orprotein.

[0044] A mutation in the L-2 cell derived HIV-1 provirus vpr geneencoding the Vpr polypeptide has also been identified. The mutation isthe substitution of A for the G at nucleotide 5194 (nucleotide 54 inFIG. 1) in a wild type vpr gene. This mutation introduces a stop codonat amino acid residue 18 such that the vpr gene encodes a truncated Vprpolypeptide containing amino acid residues 1 through 17.

[0045] The invention also provides a substantially pure nucleic acid orfragment thereof that encodes a Vpr polypeptide comprising a nucleotidesequence that encodes substantially the same sequence of amino acidresidues 1 through 17 of an HIV-1 Vpr polypeptide.

[0046] In addition to the nucleic acids described above encodingindividual Nef, Env gp41, Pol protease, Env gp120 and Vpr polypeptidemutants, the invention also provides substantially purified nucleicacids encoding combinations of these mutant polypeptides. In oneembodiment, any of the mutations in an individual polypeptide can beused alone or in combination with one or more additional mutations inthe same polypeptide. Thus, the invention provides a substantially purenucleic acid or fragment thereof that encodes a HIV-1 polypeptide havingone or more of the mutations described above.

[0047] For example, a nucleic acid encoding a Nef polypeptide caninclude the deletion mutant, which encodes amino acid residues 1 through56 of the Nef polypeptide, and any combination of one or more of theamino acid substitutions. The invention provides a nucleic acid encodingthe truncated Nef polypeptide containing amino acid residues 1 through56 and Ala at amino acid residue 15. A nucleic acid can also encode thetruncated Nef polypeptide with Lys at amino acid residue 19. Similarly,any of the amino acid substitution mutations in the Nef polypeptidedescribed above can be combined with the truncated Nef polypeptide.Alternatively, any of the amino acid substitution mutations can becombined with each other in the full length Nef polypeptide or afragment thereof.

[0048] The invention thus provides a substantially pure nucleic acid orfragment thereof that encodes at least one of the mutations describedabove and can, where more than one mutation has been described in anindividual polypeptide, contain any combination of two or moremutations, including as many as all of the mutations described for anindividual polypeptide. An example of such a nucleic acid encoding anindividual polypeptide having multiple mutations is shown in FIG. 3 (SEQID NO:45).

[0049] In another example of a nucleic acid encoding an individualpolypeptide for which multiple mutations have been identified, a nucleicacid encoding Env gp120 can contain the deletion of up to eight aminoacids of amino acid residues 144 through 151. A nucleic acid encodingEnv gp120 can also contain the deletion of one or two amino acids ofamino acid residues 192 and 193. A nucleic acid encoding Env gp120 canadditionally contain the combination of deletion of up to eight aminoacids of amino acid residues 144 through 151 and deletion of one or twoamino acids of amino acid residues 192 and 193. Additionally, anycombination of one or both of the deletion mutations can be combinedwith Arg at amino acid residue 143. The combination of one or both ofthe deletion mutations can also be combined with Ile at amino acidresidue 153. Also, the combination of one or both of the deletionmutations can be combined with Val at amino acid residue 187.Additionally, the combination of one or both of the deletion mutationscan be combined with one or two of the single amino acid substitutions.

[0050] In another embodiment in which nucleic acids encode a combinationof amino acid mutations of polypeptides, the invention also provides asubstantially pure nucleic acid or fragment thereof that encodes morethan one polypeptide containing at least one mutation. A nucleic acid ofthe invention can therefore encode any one or more of the polypeptidesselected from the group consisting of Nef, Env gp41, Pol protease, Envgp120 and Vpr polypeptides so long as the encoded polypeptides containat least one of the mutations described above. For example, asubstantially pure nucleic acid can encode a truncated Nef polypeptidecontaining amino acid residues 1 through 56 and Env gp41 containing Argat amino acid residue 660. In another embodiment, a substantially purenucleic acid can encode three or more of the polypeptides selected fromthe group consisting of Nef, Env gp41, Pol protease, Env gp120 and Vprpolypeptides, up to and including all of these polypeptides so long asat least one of the amino acid mutations described above is encoded bythe nucleic acid. For example, a substantially pure nucleic acid canencode all of the polypeptides Nef, Env gp41, Pol protease, Env gp 120and Vpr polypeptides having all of the amino acid mutations describedabove.

[0051] Further aspects of this invention are the substantially purepolypeptides or fragments thereof that are encoded by any of the abovenucleic acid molecules. In one aspect of the invention, thesubstantially pure polypeptides or fragments thereof can be used toraise antibodies which are specific to the mutated proteins.

[0052] The invention provides a substantially pure HIV-1 polypeptide ora fragment thereof selected from the group of polypeptides consisting ofNef, Env gp41, Pol protease, Env gp120 and Vpr, wherein the HIV-1polypeptide contains at least one of the mutations found in HIV-1provirus from L-2 cells. Such mutations found in HIV-1 provirus from L-2cells are described above.

[0053] In one embodiment, the invention provides Nef polypeptides havingone or more mutations in the Nef polypeptide of a HIV-1 provirus fromL-2 cells. For example, the invention provides a substantially pure Nefpolypeptide or fragment thereof comprising substantially the samesequence of amino acid residues 1 through 56 of a HIV-1 Nef polypeptide.The invention also provides a substantially pure Nef polypeptide orfragment thereof comprising substantially the same amino acid sequenceof an HIV-1 Nef polypeptide containing Ala at amino acid residue 15. Theinvention additionally provides a substantially pure Nef polypeptide orfragment thereof comprising substantially the same amino acid sequenceof an HIV-1 Nef polypeptide containing Lys at amino acid residue 19.

[0054] The invention further provides a substantially pure Nefpolypeptide or fragment thereof comprising substantially the same aminoacid sequence of an HIV-1 Nef polypeptide containing Val at amino acidresidue 33. The invention also provides a substantially pure Nefpolypeptide or fragment thereof comprising substantially the same aminoacid sequence of an HIV-1 Nef polypeptide containing Asp at amino acidresidue 54.

[0055] In addition to the Nef polypeptides having the individualmutations described above, the invention also provides a substantiallypure Nef polypeptide or fragment thereof having any combination of oneor more of the Nef polypeptide mutations. For example, the inventionprovides Nef polypeptides having one or more of the amino acidsubstitution mutations described above in the full length Nefpolypeptide. Furthermore the invention provides a truncated Nefpolypeptide and a combination of one or more of the amino acidsubstitution mutations described above. For example, the inventionprovides a truncated Nef polypeptide containing amino acids 1 through 56and Ala at amino acid residue 15. Additionally, the invention provides atruncated Nef polypeptide containing amino acids 1 through 56 and Lys atamino acid residue 19. Further provided is a truncated Nef polypeptidecontaining amino acids 1 through 56 and Val at amino acid residue 33.Also provided is a truncated Nef polypeptide containing amino acids 1through 56 and Asp at amino acid residue 54.

[0056] In addition to the Nef polypeptide truncation mutants combinedwith a single amino acid substitution mutation, the invention alsoprovides a truncated Nef polypeptide containing amino acids 1 through 56and two or more of the amino acid substitution mutations describedabove. For example, the Nef polypeptide can contain amino acid residues1 through 56 and Asp at amino acid residue as well as Val at amino acidresidue 33. Furthermore, the invention provides a truncated Nefpolypeptide containing amino acid residues 1 through 56 as well as Alaat amino acid residue 15, Lys at amino acid residue 19, Val at aminoacid residue 33, and Asp at amino acid residue 54. Therefore, one aspectof the instant invention is the truncated HIV-1 Nef protein containingthe amino acid substitutions set forth in SEQ ID NO: 46 (FIG. 4). Theamino acid sequence of such a Nef polypeptide combining all of the Nefpolypeptide mutations found in HIV-1 provirus from L-2 cells is shown inFIG. 4 (SEQ ID NO:46).

[0057] The invention also provides a substantially pure Env gp41polypeptide or fragment thereof comprising substantially the same aminoacid sequence of an HIV-1 Env gp4l polypeptide containing Arg at aminoacid residue 660. The invention additionally provides a substantiallypure Pol protease polypeptide or fragment thereof comprisingsubstantially the same sequence of amino acid residues 1 through 13 ofan HIV-1 Pol protease polypeptide fused at the carboxyl-terminus tosubstantially the same amino acid sequence asAsnAspArgGlyAlaThrLysGlySerSerIleArgTyrArgSerArg (SEQ ID NO:6).

[0058] The invention also provides a substantially pure Env gp120polypeptide or fragment thereof comprising substantially the same aminoacid sequence of an HIV-1 Env gp120 polypeptide containing Arg at aminoacid residue 143, Ile at amino acid residue 153, and Val at the aminoacid residue corresponding to amino acid residue 187 of a wild typeHIV-1 Env gp120 polypeptide and absent amino acid residues correspondingto amino acid residues 144 through 151 and 192 through 193 of a wildtype HIV-1 Env gp120 polypeptide.

[0059] The invention also provides a substantially pure Env gp120polypeptide comprising substantially the same amino acid sequence of anEnv gp120 polypeptide with deletion of at least one and up to eightamino acids of amino acid residues 144 through 151. The inventionadditionally provides a substantially pure Env gp120 polypeptidecomprising substantially the same amino acid sequence of an Env gp120polypeptide with deletion of one or both of amino acid residues 192 and193.

[0060] Additional Env gp120 polypeptides are also provided in thepresent invention. For example, the invention provides an Env gp120polypeptide comprising deletion of at least one and up to eight aminoacids of amino acid residues 144 through 151 and deletion of one or bothof amino acid residues 192 and 193. Additionally, the invention providesEnv gp120 polypeptides comprising deletion of at least one and up toeight amino acids of amino acid residues 144 through 151 and deletion ofone or both of amino acid residues 192 and 193 as well as Arg at aminoacid residue 143. The invention also provides Env gp120 polypeptidescomprising deletion of at least one and up to eight amino acids of aminoacid residues 144 through 151 and deletion of one or both of amino acidresidues 192 and 193 as well as Ile at amino acid residue 153. Also, theinvention provides Env gp120 polypeptides comprising deletion of atleast one and up to eight amino acids of amino acid residues 144 through151 and deletion of one or both of amino acid residues 192 and 193 aswell as Val at amino acid residue 187.

[0061] The invention further provides Env gp120 polypeptides comprisingdeletion of at least one and up to eight amino acids of amino acidresidues 144 through 151 and deletion of one or both of amino acidresidues 192 and 193 as well as two of the amino acid substitutionmutations. For example, the invention provides Env gp120 polypeptidescomprising deletion of at least one and up to eight amino acids of aminoacid residues 144 through 151 and deletion of one or both of amino acidresidues 192 and 193 as well as Arg at amino acid residue 143 and Ile atamino acid residue 153. The invention also provides Env gp120polypeptides comprising deletion of at least one and up to eight aminoacids of amino acid residues 144 through 151 and deletion of one or bothof amino acid residues 192 and 193 as well as Arg at amino acid residue143 and Val at amino acid residue 187. The invention further providesEnv gp120 polypeptides comprising deletion of at least one and up toeight amino acids of amino acid residues 144 through 151 and deletion ofone or both of amino acid residues 192 and 193 as well as Ile at aminoacid residue 153 and Val at amino acid residue 187.

[0062] The invention also provides a substantially pure Vpr polypeptideor fragment thereof comprising substantially the same sequence of aminoacid residues 1 through 17 of an HIV-1 Vpr polypeptide.

[0063] As discussed above for Nef polypeptide and Env gp120, any of themutations in an individual polypeptide can be used alone or incombination with one or more additional mutations in the samepolypeptide. Thus, the invention provides a substantially pure HIV-1polypeptide or fragment thereof selected from the group consisting ofNef, Env gp41, Pol protease, Env gp120 and Vpr that contain two or moreof the mutations described above in a single individual polypeptide.

[0064] The invention thus provides a substantially pure HIV-1polypeptide or fragment thereof selected from the group of polypeptidesconsisting of Nef, Env gp41, Pol protease, Env gp120 and Vpr, whereinthe HIV-1 polypeptide or fragment thereof comprises at least one of themutations described above and can, where more than one mutation has beendescribed in an individual polypeptide, comprise any combination of twoor more mutations, including as many as all of the mutations describedfor an individual polypeptide.

[0065] Any combination of one or more of the HIV-1 polypeptidescontaining any combination of one or more of the mutations describedabove can be expressed together as desired. The choice of combination ofpolypeptides and the combination of mutations in the polypeptidesdepends on the needs of the investigator.

[0066] The invention also provides nucleic acids encoding substantiallythe same amino acid sequence of the mutant HIV-1 polypeptides describedabove. Any nucleic acid that encodes any combination of single mutationsin a polypeptide, multiple mutations in a polypeptide or a combinationof two or more polypeptides of the invention are also provided.

[0067] The genes of HIV-1 provirus from L-2 cells described above wereisolated using PCR amplification as described in Example I. However, thenucleic acid molecules described above can be isolated from L-2 cells byany of a variety of methods well known in the art. For example, inaddition to PCR amplification of nucleic acids, other methods well knownin the art regarding cloning of DNA from an organism can be used toisolate HIV-1 provirus DNA (Sambrook et al., Eds., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Springs Harbor Laboratory Press (1989),which is herein incorporated by reference). Furthermore, the nucleicacids of the invention, which are set forth above, can be synthesized bychemical means (See, Blackburn and Gait, Eds., Nucleic Acids inChemistry and Biology, 2nd Ed., Oxford University Press (1996), which isherein incorporated by reference).

[0068] The proteins and peptides encoded by said nucleic acids can beisolated and purified from L-2 viral particles (see, for example,Deutscher, Ed., Methods in Enzymology: Guide to Protein Purification,Vol. 182, Academic Press, Inc., San Diego, Calif. (1990), hereinincorporated by reference), or chemically synthesized or expressed in arecombinant organism by methods well known in the art, such as Stewartand Young, Eds., Solid Phase Peptide Synthesis, 2nd Ed., Pierce ChemicalCompany (1984); Kriegler, M., Ed., Gene Transfer and Expression: ALaboratory Manual, Stockton Press, New York, N.Y. (1990); and Jones, J.,Ed., The Chemical Synthesis of Peptides, Oxford University Press, Oxford(1994), all of which are herein incorporated by reference. For example,the substantially pure nucleic acids of the invention can be expressedrecombinantly in bacterial or eukaryotic cells and subsequentlypurified.

[0069] As non-infectious particles, the L-2 viral particles can be usedas immunogens to induce a host immune response against an HIV infection.More specifically, the immunogens can be used for the prevention orreduction of apoptotic cell lysis caused by HIV infection.

[0070] The L-2 viral particles can be produced by methods well known inthe art. For example, non-infectious viral particles can be produced bygrowing L-2 cells in culture under conditions such thatprotease-defective L-2 HIV particles are produced. Such particles arethen separated from L-2 cells by, for example, centrifugation of the L-2cell supernatant and recovering the particles in the pellet. Suchmethods are described further below in the Examples as well as in, forexample, Ohki et al., J. Acquired Immun. Def. Syn. 4:1233-1240 (1991).

[0071] The non-infectious L-2 viral particles can be used directly as animmunogen or, alternatively, can be further treated to ensure multiple,additional levels of viral particle inactivation. Additionalinactivation treatment is a precautionary measure to provide furtherconfidence and safety levels against possible commination of non-L-2 HIVviral particles. Methods of viral particle inactivation are well knownin the art and include, for example, treatment with gamma-radiation,beta-propiolactone and gluaraldehye. These methods are described furtherbelow in the Examples. Moreover, the production and use of immunogens istaught, for example, in Salk et al., U.S. Pat. No. 5,256,767, issuedOct. 26, 1993. Therefore, the invention provides an immunogen consistingof non-infectious, protease-defective particles produced by L-2 cells.The invention also provides inactivated, protease-defective particlesproduced by L-2 cells. The non-infectious, protease-defective particlescan also be produced by non-L-2 cells which harbor the L-2 provirusgenome.

[0072] As described above, the L-2 viral particles contain a number ofmutations which render the particles non-infectious. These mutations canbe advantageously exploited to engineer a variety of non-infectiousviral particles as specific immunogens against preselected types of HIVviruses. For example, it is not necessary to have all of the mutationsdescribed herein for the particle to be non-infectious and thereforeuseful as an immunogen. Instead, immunologically effective immunogenscan be prepared from modified forms of the L-2 viral particlescontaining less than all of the mutations described herein so long asthe modified L-2 viral particle contains a mutation that renders theparticle non-infectious. Such mutations can be, for example, any one ofthe mutations identified in the Pol protease, Nef, or Env gp41, forexample. These mutations can be combined with the L-2 Env gp120polypeptide or the L-2 Env gp120 mutants described herein to producenon-infectious viral particles for vaccinating against HIV. Other mutantpolypeptides such as the L-2 Vpr or the L-2 Vpr mutants described hereincan also be included within the viral particles to augment thenon-infectious phenotype of the modified L-2 viral particles. Thoseskilled in the art will know or can determine which mutation orcombination of mutations are sufficient to render the particlenon-infections given the mutant sequences and teachings describedherein.

[0073] Alternatively, the L-2 mutations identified in the Pol protease,Nef, or Env gp41 polypeptides can be combined with Env gp120polypeptides from other HIV virus sub-types, or clades, to producenon-infectious viral particles. These particles can similarly be used asvaccines against the various HIV clades. For example, there are twoprimary groups of HIV clades, termed M and O, which characterize theinfective phenotype of the virus. For group M, there are eight clades (Athrough J). The L-2 viral particles are characterized as being withinthe B clade of Group M, Betts et al., J. Virology 71:8909-8911 (1997).

[0074] The L-2 viral particle are characterized as being within the Oclade having subgroup E. Substituting Env gp120 derived from differentclades or clade subgroups into non-infectious L-2 viral particles willrender a modified L-2 particle specific for the substituted Env gp120clade. These clade-specific, modified L-2 particles can then be used asimmunogens for the prevention and treatment of an HIV infection.

[0075] As with the modified L-2 viral particles described above,immunogenically effective immunogens can be prepared from theclade-specific forms of the modified L-2 viral particles that containless than all of the L-2 mutations described herein so long as theparticles contain at least one of the mutations that renders theparticle non-infectious. Such mutations can be, for example, any one ofthe mutations identified in the Pol protease, Nef, or Env gp41, forexample. Additionally, the mutations described in the Vpr protein can beoptionally incorporated. Those skilled in the art will know or candetermine which mutation or combination of mutations are sufficient torender the clade-specific viral particles non-infections given themutant sequences and teachings described herein.

[0076] Production of the modified L-2 particles or the modified,clade-specific particles described above can be performed using methodswell known in the art given the L-2 nucleotide and amino acid sequencesand the teachings described herein. Such methods can include, forexample, the construction and expression of recombinant modified L-2genomes so as to contain a desired combination of the L-2 mutationswhich confer the non-infectious phenotype. The construction of suchdesired combinations can be performed by, for example, site directedmutagenesis or by polymerase chain reaction (PCR) using mutagenicprimers. Other well known methods exist in the art and can similarly beused to generate the desired combination of L-2 mutations within toproduce a modified L-2 genome or a modified, clade specific genome. Onceproduced, the genomes can be stably introduced into compatible cells,such as T cells, to harbor the provirus and effect the production of themodified viral particles. Such methods are well known in the art and canbe found described in, for example, Sambrook et al., Eds., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989), and Ausubel et al., Eds., Current Protocols in MolecularBiology, John Wiley & Sons, (1987). The non-infectious, modified L-2particles, or the non-infectious, modified particles that exhibitclade-specific phenotypes can be isolated and used directly asimmunogens or be subjected to further inactivation procedures asdescribed above prior to use as immunogens for the prevention ortreatment of apoptotic cell lysis mediated by HIV.

[0077] Specific examples of non-infectious, modified L-2 viral particlesinclude particles containing at least an expressible Nef or Pol proteasemutant as described previously. For example, the Nef mutant can containthe stop codon at position 57 and/or contain one or more of thenucleotide and corresponding amino acid mutations set forth in FIGS. 3and 4, (SEQ ID NOS:45 and 46, respectively). The Pol protease mutant cancontain one or more of the nucleotide and corresponding amino acidmutations set forth in FIG. 1 (SEQ ID NO:5 and 6, respectively). Any ofthe Nef or Pol protease mutants can additionally be combined with thevarious Vpr, Env gp41 or Env gp120 mutants previously describe togenerate a non-infectious, modified L-2 viral particle of the invention.Similarly, such mutations can be constructed with non-L-2 Env gp120polypeptides to produce non-infectious, modified L-2 particles that areclade specific.

[0078] Any of the previously described non-infectious L-2 viral particleimmunogens, including modified forms and clade-specific forms, canadditionally be combined with other HIV immunogens to further augment animmune response directed to HIV. Additional HIV immunogens can include,for example, inactivated forms of the virus, virus which is devoid ofits outer envelope proteins, as well as soluble HIV polypeptides orfragments thereof. These additional HIV immunogens that can be combinedwith the non-infectious L-2 particles and modified forms thereof, can bederived from HIV-1, HIV-2 or both, for example. Such combined immunogenscan similarly be formulated in an adjuvant for administration.Immunogens for the induction of an immune response against HIV are wellknown in the art. A specific example of an immunogen which is devoid ofthe Env gp120 polypeptide is described, for example, in Salk et al.,supra.

[0079] Therefore, the invention provides a method for the reduction orprevention of apoptotic cell lysis mediated by HIV. The method consistsof administration of a non-infectious L-2 particle or non-infectiousmodified L-2 particle to a human who is either seropositive orseronegative for HIV, and particularly in those patients where theapoptotic lysis is induced by protease-defective particles.Additionally, the invention provides for methods of stimulating theimmune system of a human. Such a method would comprise administering oneof the above non-infectious L-2 particle or modified L-2 particleimmunogens, or a composition including such immunogens in animmunologically effective amount to a human. Again, the human could beseronegative or seropositive for HIV.

[0080] The non-infectious immunogens of the invention are administeredto a host exhibiting or at risk of exhibiting an autoimmune response.Definite clinical diagnosis of AIDS or AIDS related complex (ARC)warrants the administration of the relevant immunogen. Prophylacticapplications are warranted where an HIV infection precedes the onset ofovert clinical disease or where the individual has been at risk ofexposure to the virus. Thus, individuals with any history of infectionor predicted to be at risk by reliable prognostic indicators can betreated prophylactically to interdict immunodeficiency mechanisms priorto their onset.

[0081] The non-infectious immunogens of the invention can beadministered in many possible formulations, including pharmaceuticallyacceptable mediums. The immunogens can include or be administered inconjunction with an adjuvant, of which several are known to thoseskilled in the art. After initial immunization with the non-infectiousimmunogen, further boosters can additionally be provided. Thenon-infectious immunogens are administered by conventional methods, indosages which are sufficient to elicit an immunological response. Suchdosages are known and also can be easily determined by those skilled inthe art.

[0082] Briefly, individuals who are candidates for immunization can beeffectively treated by active immunotherapy using a non-infectiousimmunogen prepared from the L2-viral particles or modified formsthereof. The dose is selected so as to be immunologically effective, andis generally between about 1 to about 100 μg of protein, more preferablyabout 30 μg of protein.

[0083] Active immunization is implemented and preferably repeated onceat a minimum interval of at least 90 days, although additional boostsmay be appropriate according to changes in the immunocompetence level,based, for example, on a decline in antibodies to HIV gene productsother than outer envelope proteins. Such immunization is preferablyaccomplished initially by intramuscular injection followed byintradermal injection, although any combination of intradermal andintramuscular injections may be used.

[0084] Preferably, the immunoresponsiveness or immunocompetence of theseropositive individual is determined prior to immunization in order todetermine an appropriate course of therapy. As a method of suchdetermination, individuals' sera can be screened for the presence ofantibodies to p24 (as by means of ELISA), for TRI antibody and/or forthe level of T₄ cells by methods well known in the art. Individualsexhibiting indicators of low immunocompetence, such as low p24 or RTIantibody titers or low numbers of T₄ cells, are appropriate candidatesfor active immunotherapy. Additionally, active immunotherapy can becombined with other therapies known in the art including, for example,passive immunotherapy.

[0085] Seronegative individuals can be vaccinated in order to induceimmunoprotective factors to prevent infection. Preferably, the vaccineis administered initially by intramuscular injection followed by abooster injection given either intramuscularly or intradermally. Aphysiologically effective dose, preferably in the range of 1 to 100 μgand more preferably about 30 μg of immunogen is provided per dose. Thevaccine can be administered in conjunction with an adjuvant, such as awater-in-oil type adjuvant. Various appropriate adjuvants are well knownin the art as reviewed by Warren and Chedid, CRC Critical Reviews inImmunology 8:83 (1988).

[0086] These methods as well as other modes of administration are wellknown in the art and can be used for the prevention or treatment of anHIV infection using the non-infectious immunogens described herein.

[0087] The nucleic acid molecules discussed above can be used as nucleicacid probes or standards in either diagnostic or in PCR procedures. Suchdiagnostic methods can be used to detect the previously described mutantDNA sequences in cells and extracellular fluids. Methods of detection byhybridization with nucleic acid probes are well known in the art and aredescribed in, for example, Hames and Higgins, Eds., Nucleic AcidHybridisation: A Practical Approach, Oxford University Press, Oxford(1991), and Innis et al., Eds., PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc. (1990). Various formats known in theart can be used for detection of L-2 specific mutations by nucleic acidhybridization. The choice of such formats will vary depending on theparticular application and need of the diagnostic procedure. Formatsinclude, for example, solid-phase hybridization and solutionhybridization. Specific examples of solid-phase hybridization formatsfor diagnostic procedures include the use of diagnostic chips containingtwo-dimensional arrays of oligonucleotides, multi-well formats such asan ELISA and blot hybridization procedures. Such formats and variouspermutations thereof are well known in the art.

[0088] Similarly, the polypeptides above could be used as assaystandards in immunoassays kits, and could be employed to raiseantibodies for immunoassays to detect the above mutated polypeptides andorganisms containing them. Methods for raising monoclonal and polyclonalantibodies specific to such polypeptides are well known in the art.(See, for example, Harlow and Lane, Eds., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory (1988). Moreover, the productionof monoclonal antibodies is described in detail in U.S. Pat. Nos. RE32011 and 4,411,993. Antibodies to L-2 mutant polypeptides canadditionally be generated using combinatorial methods well known in theart. Such methods include the generation and screening of recombinantantibody library repertoires for antibodies specific to an antigen ofinterest, including any of the L-2 mutant polypeptides described herein(see, for example, Huse et al., Science 246:1275-1281 (1989)).Monoclonal antibodies specific for the L-2 polypeptide described hereincan additionally be modified by recombinant methods known in the art toincorporate human antibody framework sequences. Such methods are wellknown in the art and are described in, for example, U.S. Pat. No.5,585,089 to Queen et al.

[0089] Once suitable antibodies or binding proteins have been obtained,they may be isolated or purified by many techniques well known to thoseof ordinary skill in the art (see Antibodies: A Laboratory Manual,Harlow and Lane (eds.), supra). Suitable techniques include peptide orprotein affinity columns, HPLC or RP-HPLC, purification on protein A orprotein G columns, or any combination of these techniques.

[0090] Therefore, the invention provides monoclonal and polyclonalantibodies, and fragments thereof specific for L-2 mutant polypeptides,as well as chimeric and humanized antibodies and fragments specific forsuch polypeptides. Such antibodies can be additionally modified with,for example, a label or a therapeutic agent for the diagnosis ortreatment of HIV.

[0091] The antibodies raised against the above-described L-2 proteinscan be used in quantitative, semi-qualitative, or qualitative diagnosticassays. Such assays, for example, can be in RIA, IRMA, and ELISAformats. Examples of such formats include a competitive or sandwicharrangement. See, for example, David et al., U.S. Pat. No.4,486,530,issued Dec. 4, 1984; Motagnier et al., U.S. Pat. No. 4,708,818, issuedNov. 24, 1987; Montagnier et al., U.S. Pat. No. 5,135,864, issued Aug.4, 1993; Gallo et al., U.S. Pat. No. 4,647,773, issued Mar. 3, 1987;Gallo et al., U.S. Pat. No. 4,520,113, issued May 28, 1985; and Gallo etal., U.S. Pat. No. 4,652,599, issued Mar. 24, 1987, all of which areherein incorporated by reference. In a competitive format, the instantantibodies are incubated with the sample to be analyzed and a measuredquantity of labeled, competing analyte. The antibody in competitiveassay is usually bound to a solid support, or is capable of being boundto such a support, as discussed below. Such binding to a solid supportcan take place before or after the instant antibody is incubated withthe sample suspected of containing one of the instant proteins. In asandwich format, two antibodies, each specific for the analyte, arereacted one at a time or simultaneously with the sample to be analyzed.Typically, one of the instant antibodies will be either be labeled witha detection means or capable of being so and the other will either beattached to a solid support or capable of being so. After theantibody-analyte-antibody sandwich complex is formed, the detectionmeans is measured. Alternatively, after the complex is formed, thecomplex is then attached to a solid support or to a detection means, orboth.

[0092] Binding to the solid support can be accomplished by an antibodythat in turn binds to one of the antibodies of the complex, the naturalor covalent attachment of the unlabeled antibody to the support, or bythe reaction of another ligand-receptor pair, such as biotin-avidin. Inthe latter case, biotin is bound either to the constant region of theantibody or to the solid support and avidin is bound to the remainingcomponent. In either the competitive or sandwich format, there areusually wash, aspiration and possibly filter procedures after theincubation of the sample and following the additions of each reagent.

[0093] The antibodies used in the above diagnostic assays can use, forexample, one or more of the instant polyclonal or monoclonal antibodies,mixtures thereof, and the corresponding functional fragments. Forexample, in a sandwich assay, the two antibodies used to form thecomplex can either both be polyclonal or monoclonal or functionalfragments thereof. Alternatively, one monoclonal and one polyclonal canform the sandwich. Similarly, in the competitive format, the antibodycan be one or more monoclonal or polyclonal antibodies, or functionalfragments thereof.

[0094] The detection means are well known in the art. Such detectionmeans can be, for example, enzymes, radioisotopes, fluorogens,chromogens, metallic or non-metallic colloids, colored liposomes,colored or colorable particles, and the like. Thus, the detection meansmay be immediately visible after the analyte complexes with the instantantibodies in an assay, or further steps may need to be taken to utilizethe detection means. Such steps include UV and visible spectrophtometry,fluorimetry and analysis by radiation counters. Examples of suchdetection means include alkaline phosphatase/para-nitrophenyl phosphate;horse radish peroxidase/aminoethylcarbazole, colloidal gold, colloidalsilver, colloidal selenium, hydrophobic dyes, colored latexmicroparticles, carbon sols, rhodamine, fluorescein, luminol, luciferin,umbeliferone, ¹²⁵Iodine, ¹³¹Iodine, tritium, ³²Phosporous, and the like.These detection means can be coupled to the either the analyte(competitive assays) or to an antibody (sandwich assays) by means wellknown in the art. Suitable detection means and methods of making themare further discussed, for example, in Tom et al., U.S. Pat. No.4,366,241, issued Dec. 28, 1982; and in Campbell, U.S. Pat. No.4,703,017, issued Oct. 27, 1987.

[0095] The optional solid supports for the assays can be bibulous ornonbibulous. Examples of bibulous supports include nitrocellulose,nylon, filter paper, and the like. Non-bibulous supports are ones thatdo not necessarily effect a chromatographic separation of the componentsas they advance along the solid support. Examples of such non-bibuloussupports include plastic materials such as high density polyethylene,polystyrene, polyvinyl chloride, mixtures of such high density plastics,or bibulous material rendered non-bibulous by the application ofnon-blocking materials, such as detergents and proteins. These and othersupports and methods of use are known in the art.

[0096] The invention also provides diagnostic kits comprising the L-2specific antibodies. In addition, such kits can contain amounts ofreagents for use in assaying for an analyte. Such reagents can besubstrates when enzymes are used as the detection means, as well asancillary reagents like stabilizers, extraction reagents, buffers, washsolutions, and the like. The relative amounts of the various reagentsvary widely, to provide for concentrations in solution of the reagentswhich substantially optimize the sensitivity of the assay. The reagentscan be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing the assay. The kitcan also be contained in packaging material, such as air-tight foil, orvarious external containers known in the art. Such external containerscan contain the device, reagents, and the instructions for use of theassays.

[0097] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I

[0098] This Example describes the determination of the genetic makeup ofthe HIV-1 provirus in L-2 cells.

[0099] In initial studies the cleavage pattern of the HIV-1 Gagpolypeptide expressed in Escherichia coli transformed with recombinantL-2 gag containing an HIV-1 wild-type pol protease gene was found to besimilar to that of parental HIV-1 (LAI) particles. Therefore, the L-2cell provirus was sequenced at other regions of the genome. Otherregions of the L-2 cell proviruses genome that were sequenced includedpol protease; env gp120 (SU) and gp41 (TM); and the accessory regiongenes such as vif, vpu, vpr, and nef. The control was the provirus inMOLT-4 cells (ATCC) persistently infected with HIV-1 (LAI), which wasdesignated as MO/LAI was used as a control.

[0100] The polymerase chain reaction (PCR) amplification of totalcellular DNA extracted from L-2 and MO/LAI was performed as previouslydescribed (Kameoka et al., Virus Genes, 12:117-129 (1996), utilizing 30cycles of 1 min at 94° C., 4 min at 60° C., followed by a finalpolymerization step for 10 min at 72° C., with a GeneAmp XL PCR kit(Perkin-Elmer Corporation, Foster City, Calif.). The primers used ateach region are summarized in FIG. 2 (Adachi et al., J. Virol,59:284-291 (1986). The PCR products were separated by agarose gelelectrophoresis and visualized by ethidium bromide staining. Theexpected bands were ligated into a pBluescript II SK(-) vector(Stratagene Cloning Systems, La Jolla, Calif.). Nucleotide sequencing ofthe amplified DNA segments was carried out using (γ-³²p)ATP-labeled T3,T7, or synthetic primers by cycle sequencing according to the protocoldescribed for the CircumVent thermal cycle sequencing kit (New EnglandBioLabs, Beverly, Mass.).

[0101] The nucleotide sequences of provirus genes in the L-2 cells werecompared with those determined from MO/LAI as well as the reported HIV-1(LAI) sequences (GenBank accession number K02013)(Wain-Hobson et al.,Cell, 40:9-17 (1985),(see FIG. 1). Comparison of the nucleotidesequences of provirus genes to the nucleotide sequences HIV-1, LAI andHIV-1 MO/LAI genes reveals significant mutations in the pol protease,env gp41, and nef genes for L-2 proviral DNA. Specifically, the proteasegene had an insertion mutation of T at nucleotide position 42, resultingin the appearance of a stop codon at amino acid residue 30. Thisexplains the protease-defective nature of L-2 particles, as evidenced byan inability to cleave the Pr55^(gag) precursor inside L-2 cells. Forthe TM envelope glycoprotein, gp41, a 1-base substitution was found atnucleotide 7781 (A to G; corresponding to nucleotide 1979 in FIG. 1),resulting in an amino acid substitution at amino acid residue 660, froma lysine to an arginine. The nef gene also had a nonsense mutation atnucleotide 8559 (G to A; corresponding to nucleotide 170 in FIG. 1),resulting in the appearance of a stop codon at amino acid residue 57, aswell as other mutations set forth in FIG. 3. All three of these changesin the protease, gp41, and nef genes were specific for L-2 proviral DNA.

[0102] In contrast, the vpr gene was similarly truncated for both L-2and MO/LAI proviral DNAs owing to a 1-base substitution at nucleotide5194 (G to A; corresponding to nucleotide 54 in FIG. 1), resulting inthe appearance of a stop codon at amino acid residue 18. The vif and vpugenes were identical for both L-2 and MO/LAI proviral DNAs. Also, thesequencing of env gp120 revealed no apparent differences between L-2 andMO/LAI although both showed V1 and V2 domains that contained deletionsof either eight or two amino acids, respectively, as compared to thereported HIV-1 (LAI) sequence. Therefore it is possible that thesedeletions in gp120 could have occurred during the long-term maintenanceof HIV-1 (LAI) in different T cell lines during the instant studies,with no apparent effect on their infectivity.

[0103] The data presented here point to the specific involvement of oneor more mutation(s) in the L-2 provirus pol protease, env gp41, and/ornef genes as the cause for the production of defective, but highlysyncytium- and apoptosis-inducing particles. In particular, the aminoacid mutation at Env gp4l accounts for the higher functional activity ofprotease-defective particles. Furthermore, the gp120 polypeptidedeletions of 8 and 2 amino acids corresponding to SEQ ID NOS:18 and 24(FIG. 1), respectively, are critical in defining the pathogenicity ofL-2 particles in the context of the L-2 Nef, protease, gp41, or Vprmutant-derived polypeptides. These L-2 particles are responsible forcausing the highly pathogenic end-stages of HIV-1 disease leading to ARCand AIDS. Thus, it is important to stimulate the immune system to attackany defective L-2 particles that accumulate during infection. The use ofinactivated L-2 particles, optionally in conjunction with other HIV-1 orHIV-2 immunogens, or both, nucleic acid sequences and fragments,polypeptides and fragments thereof, and antibodies, as described in thisinvention, alone or in combination thereof, will remove potentiallyharmful defective L-2 particle from HIV-1 infected tissues ofindividuals, leading to an improved outcome.

[0104] These results show that HIV-1 provirus from L-2 cells havemutations in the pol protease, nef, env and vpr genes that encode thepol protease, Nef, gp41, gp120 and Vpr polypeptides and result inmutations in these polypeptides.

EXAMPLE II Production of Non-infectious, L-2 Particles or Modified FormsThereof

[0105] This Example describes the production of non-infectious L-2particles or modified forms thereof. The method of production ofnon-infectious, L-2 particles or modified forms thereof has essentiallybeen described previously with some modifications (Prior et al., (1995);Prior et al. (1996)). The modifications are related to the cell cultureand downstream recovery conditions to improve both production levels andpurification of virus from the growth medium containing 5-10% fetalbovine serum.

[0106] The production method for L-2 particles will mimic those used forHIV-1 described below. The initial production steps involve anionexchange chromatography. Briefly, the cell line used is the L-2 cellline. After multiple cell culture expansions with cell densities in therange of one to two million cells per milliliter, the cellular debris isseparated from virus by dead-end filtration using a 1.2 μM celluloseester membrane. The filtered supernatant containing L-2 particles isinactivated by addition of β-propiolactone (1:2000 v/v), concentrated bytangential flow ultrafiltration using a 300,000 molecular weight (MW)cut-off polysulphone membrane and the concentrates are stored at −70° C.This hold step permits analysis of in-process material in an infectivityassay to determine that infectious virus is nondetectable before furtherprocessing. Thawed concentrates are pooled, filtered and applied to TMAEFractogel (Merck, Darmstadt, Germany), washed with 0.5M NaCl at pH 6.5and eluted in 1.0M Ncl at pH 6.5. After immediate dilution to reduceNaCl concentration to about 0.15M, product is applied to Q-Sepharose(Pharmacia, Inc., Piscataway, N.J., USA), washed in 0.7M NaCl and elutedin 1.OM NaCl, all at pH 6.5. Following concentration/diafiltration(polysulphone, 3000 MW cut-off), product is centrifuged and the pelletis resuspended, then frozen and subjected to γ irradiation on dry ice.The combined inactivation steps, β-propiolactone and γ irradiation, canbe validated to attain a >17 log viral inactivation capability.

[0107] Briefly, the pooled filtrate containing product is inactivatedwith β-propiolactone (βNPL) and subjected to ultrafiltration. Briefly,following pH adjustment of the pooled filtrate to 7.3±0.1, βPPL is addedwhile stirring to a final concentration of 1:2,000 (v/v). After stirringfor one hour, the preparation is transferred to a second mixing vesselto ensure complete viral inactivation and incubated at 4° C. for 18-24hours with continuous stirring. The temperature of the filtrate israised to 37° C. and maintained at that temperature for approximatelyfive hours to hydrolyze residual βPL into an isomer of lactate andbetapropionic acid derivatives. Approximately 10-200 liters of postβPL-treated filtrate is concentrated to 2.5-3.5 liters using apolysulfone 300,000 molecular weight (MW) cutoff membrane anddiafiltered against 10-20 volumes of phosphate buffered saline. Thisstep removes more than 95% of the growth medium-derived polypeptide fromthe permeate, including approximately 50% of the starting p24 antigen asmeasured by ELISA. In this MW fraction (less than 300 kDa), p24 antigenis not associated with intact particles. The MW cutoff properties of themembrane achieves almost total retention of intact particles. Theconcentrate is stored frozen at −70° C. This is a critical hold stepbecause individual concentrates, derived from a single expansion of cellculture, can be thawed and pooled to the required lot size forsubsequent chromatography. This hold step can be used for qualitycontrol to determine which concentrates to release for furtherprocessing, after conducting sterility testing and after verifying thata cell culture is free of adventitious virus and mycoplasma. Thisin-process check before pooling can prevent compromising large lotsizes, thus avoiding economic risk while maintaining the safety andintegrity of a process.

[0108] Validating the use of βPL for inactivating HIV-1 is anothercritical safety measure. To determine the effectiveness of inactivatingHIV-1 with βPL, a high concentrate of infectious virus is used at 0 time(starting time, pre-βPL). βPL is added (1:2,000 v/v), and the decay ininfectivity and βPL is evaluated. After adding βPL, samples are takenhourly, βPL activity is immediately neutralized with sodium sulfite, andthe material is maintained at −70° C. pending analysis of theinfectivity and βPL assays. Evaluation of inactivation is computed bysubtracting the final log concentration at the time point measured fromthe log concentration of infectious HIV-1 at 0 time (pre-βPL). Previousunpublished work has shown that, within 12 hours, infectious virus iseither nondetectable or at trace levels, and absolute inactivation isconfirmed 18 hours after βDPL is added. Because the half-life of βPL inproduction medium is approximately 19 hours, sufficient residual βPLexists to continue inactivation beyond the 12-18 hours during whichinfectious virus is nondetectable. Therefore, to evaluate theinactivation potential for the entire production time of ≦53 hours, afresh concentration of infectious HIV-1 is added in the form of aninfectious spike to the βPL-treated material after 12 hours to determinethe cumulative inactivation. Separate validation runs can performed induplicate.

[0109] For large scale cell culture and harvest of L-2 particles, thecell line used is L-2 cells. Briefly, frozen vials of L-2 cells arethawed and cultures are initiated. The culture medium throughout most ofthe expansion process is 10% fetal bovine serum (FBS) in RPM1-1640 with25 mM HEPES - N-(2-hydroxyethyl)piperazine-N′-2-ethanesulphonic acid. Asthe cells divide, the culture volume is expanded to larger vessels andsparged daily with 5% CO₂/air. In the final expansion (fromapproximately 72L to a final volume of 144 L), the FBS content isreduced to about 7.5% with 5.0% FBS in RPM1-1640 and 25 nM HEPES. Virusis separated from intact cells and cellular debris using a 1.2-μmcellulose ester filter. The pooled filtrate-containing product is cooledand maintained at 4° C. The entire cell culture production cycle, fromthe initial thaw of cells to the harvest of the final expansion takesapproximately 35-45 days.

[0110] For cobalt irradiation and final formulation, the frozen,postchromatography product is removed from a biosafety level 3 (BSL-3)containment facility and on dry ice is subjected to 4.5-5.5 Mrad ofcobalt irradiation. The objective is to optimize virus inactivationpotential and minimize alteration to polypeptide structure to produce animmunogen. Cobalt irradiation inactivates virus by shearing nucleic acidinto pieces of approximately 200 base pairs (bp), as determined bypolymerase chain reaction (PCR), rendering virus biologicallydysfunctional. The HIV-1 inactivation efficiency of cobalt irradiationis evaluated by aliquoting a given preparation of starting high-tierinfectious virus and subjecting each sample maintained at −70° C. to asingle dose level of irradiation. Individual alquots are subjected to1.0, 2.0, 3.0, 4.0, 5.0 Mrad and measured for levels of infectivity. Thestudy is performed in triplicate using three separate lots of infectiousvirus. The log reduction of infectivity is determined in primaryperipheral blood mononuclear cells using the starting infectious titerfollowing 5 Mrads of irradiation. The mean reduction in infectivity isexpected to be 7 logs.

[0111] Cobalt irradiation also functions to sterilize the product. Thisis important because, unlike polypeptide solutions, purified HIV-1particles such as non-infectious, L-2 particles or modified formsthereof cannot be subjected to sterile filtration techniques. A 0.2 μmfilter retains HIV-1 particles. Following irradiation, product isthawed, diluted with saline to a required dose, and emulsified with anequal volume of incomplete Freund's adjuvant. Syringes are filled forintramuscular administration with final bulk product—an off-white,viscous emulsion. Consistent sterility of product is maintained byperforming formulation steps in an aseptic area supplied with highquality air such as class 100 filtered air maintained under positivepressure.

[0112] Inactivated purified product is thawed, polypeptide contentdetermined, diluted in saline and emulsified with an equal volume ofincomplete Freund's adjuvant (IFA) (Seppic Inc. Paris, France), toachieve a water:oil ratio of 1:1. All liquids and containers aremaintained at 2-8° C. to attain an optimal viscosity. The bulk productis filled into syringes for intramuscular administration.

[0113] To determine the purity of the final viral particle product,analytical gel-filtration chromatography is used. Analyticalgel-filtration chromatography CL6B resin (Pharmacia Inc.) is packed intoa 16 mm×310 mm low-pressure Pharmacia XK16 column. The total columnvolume is about 60 ml. Low-pressure chromatography conditions areemployed during the separation procedure to ensure that the virionremains intact, including use of a non-denaturing buffer system. Therunning buffer is 0.1M Tris and 0.5M NaCl, pH 8.0. This ionic strengtheliminates any adsorption of the virion onto the resin. The CL6B resinis highly crosslinked and has a MW fractionation range between 10 and4000 kDa (molecules with a MW>4000 kDa are eluted in the void volume).The column is controlled by a Perspective Biosystems BioCAD 60 controlsystem (Cambridge, Mass. USA) at a flow rate of 2 ml min.Chromatographic profiles are monitored at two wavelengths: 230 and 280nm. To determine HIV-1 antigen recovery and calculate mass balance,approximately 1.5 ml fractions are collected.

[0114] The non-infectious, L-2 particles or modified forms thereofproduced by the procedure described above can be used for the preventionor treatment of diseases caused by HIV infection.

[0115] Throughout this application various publications have beenreferenced within parentheses. The disclosures of these publications intheir entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

[0116] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 48 1 51 DNA Human immunodeficiency virus type 1 CDS (1)..(51) 1 aagata ggg ggg caa cta aag gaa gct cta tta gat aca gga gca gat 48 Lys IleGly Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala Asp 1 5 10 15 gat 51Asp 2 17 PRT Human immunodeficiency virus type 1 2 Lys Ile Gly Gly GlnLeu Lys Glu Ala Leu Leu Asp Thr Gly Ala Asp 1 5 10 15 Asp 3 51 DNA Humanimmunodeficiency virus type 1 CDS (1)..(51) 3 aag ata ggg ggg caa ctaaag gaa gct cta tta gat aca gga gca gat 48 Lys Ile Gly Gly Gln Leu LysGlu Ala Leu Leu Asp Thr Gly Ala Asp 1 5 10 15 gat 51 Asp 4 17 PRT Humanimmunodeficiency virus type 1 4 Lys Ile Gly Gly Gln Leu Lys Glu Ala LeuLeu Asp Thr Gly Ala Asp 1 5 10 15 Asp 5 52 DNA Human immunodeficiencyvirus type 1 CDS (1)..(51) 5 aat gat agg ggg gca act aaa gga agc tct attaga tac agg agc aga 48 Asn Asp Arg Gly Ala Thr Lys Gly Ser Ser Ile ArgTyr Arg Ser Arg 1 5 10 15 tga t 52 6 16 PRT Human immunodeficiency virustype 1 6 Asn Asp Arg Gly Ala Thr Lys Gly Ser Ser Ile Arg Tyr Arg Ser Arg1 5 10 15 7 60 DNA Human immunodeficiency virus type 1 CDS (1)..(60) 7atg gaa caa gcc cca gaa gac caa ggg cca cag agg gag cca cac aat 48 MetGlu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu Pro His Asn 1 5 10 15gaa tgg aca cta 60 Glu Trp Thr Leu 20 8 20 PRT Human immunodeficiencyvirus type 1 8 Met Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln Arg Glu ProHis Asn 1 5 10 15 Glu Trp Thr Leu 20 9 60 DNA Human immunodeficiencyvirus type 1 CDS (1)..(54) 9 atg gaa caa gcc cca gaa gac caa ggg cca cagagg gag cca cac aat 48 Met Glu Gln Ala Pro Glu Asp Gln Gly Pro Gln ArgGlu Pro His Asn 1 5 10 15 gaa tga acacta 60 Glu 10 17 PRT Humanimmunodeficiency virus type 1 10 Met Glu Gln Ala Pro Glu Asp Gln Gly ProGln Arg Glu Pro His Asn 1 5 10 15 Glu 11 60 DNA Human immunodeficiencyvirus type 1 CDS (1)..(54) 11 atg gaa caa gcc cca gaa gac caa ggg ccacag agg gag cca cac aat 48 Met Glu Gln Ala Pro Glu Asp Gln Gly Pro GlnArg Glu Pro His Asn 1 5 10 15 gaa tga acacta 60 Glu 12 17 PRT Humanimmunodeficiency virus type 1 12 Met Glu Gln Ala Pro Glu Asp Gln Gly ProGln Arg Glu Pro His Asn 1 5 10 15 Glu 13 36 DNA Human immunodeficiencyvirus type 1 CDS (1)..(36) 13 agt agt aat acc aat agt agt agc ggg gaaatg atg 36 Ser Ser Asn Thr Asn Ser Ser Ser Gly Glu Met Met 1 5 10 14 12PRT Human immunodeficiency virus type 1 14 Ser Ser Asn Thr Asn Ser SerSer Gly Glu Met Met 1 5 10 15 12 DNA Human immunodeficiency virus type 1misc_feature (4)..(6) any nucleotides that code for Arg. 15 agtmgnatgata 12 16 4 PRT Human immunodeficiency virus type 1 16 Ser Arg Met Ile 117 12 DNA Human immunodeficiency virus type 1 misc_feature (4)..(6) anynucleotides coding for Arg 17 agtmgnatga ta 12 18 4 PRT Humanimmunodeficiency virus type 1 18 Ser Arg Met Ile 1 19 27 DNA Humanimmunodeficiency virus type 1 CDS (1)..(27) 19 ata ata cca ata gat aatgat act acc 27 Ile Ile Pro Ile Asp Asn Asp Thr Thr 1 5 20 9 PRT Humanimmunodeficiency virus type 1 20 Ile Ile Pro Ile Asp Asn Asp Thr Thr 1 521 21 DNA Human immunodeficiency virus type 1 CDS (1)..(21) 21 ata gtacca ata gat aat acc 21 Ile Val Pro Ile Asp Asn Thr 1 5 22 7 PRT Humanimmunodeficiency virus type 1 22 Ile Val Pro Ile Asp Asn Thr 1 5 23 21DNA Human immunodeficiency virus type 1 CDS (1)..(21) 23 ata gta cca atagat aat acc 21 Ile Val Pro Ile Asp Asn Thr 1 5 24 7 PRT Humanimmunodeficiency virus type 1 24 Ile Val Pro Ile Asp Asn Thr 1 5 25 39DNA Human immunodeficiency virus type 1 CDS (1)..(39) 25 att gaa gaa tcgcaa aac cag caa gaa aag aat gaa caa 39 Ile Glu Glu Ser Gln Asn Gln GlnGlu Lys Asn Glu Gln 1 5 10 26 13 PRT Human immunodeficiency virus type 126 Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln 1 5 10 27 39 DNAHuman immunodeficiency virus type 1 CDS (1)..(39) 27 att gaa gaa tcg caaaac cag caa gaa aag aat gaa caa 39 Ile Glu Glu Ser Gln Asn Gln Gln GluLys Asn Glu Gln 1 5 10 28 13 PRT Human immunodeficiency virus type 1 28Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln 1 5 10 29 39 DNAHuman immunodeficiency virus type 1 CDS (1)..(39) 29 att gaa gaa tcg caaaac cag caa gaa agg aat gaa caa 39 Ile Glu Glu Ser Gln Asn Gln Gln GluArg Asn Glu Gln 1 5 10 30 13 PRT Human immunodeficiency virus type 1 30Ile Glu Glu Ser Gln Asn Gln Gln Glu Arg Asn Glu Gln 1 5 10 31 51 DNAHuman immunodeficiency virus type 1 CDS (1)..(51) 31 aca agt agc aat acagca gct acc aat gct gct tgt gcc tgg cta gaa 48 Thr Ser Ser Asn Thr AlaAla Thr Asn Ala Ala Cys Ala Trp Leu Glu 1 5 10 15 gca 51 Ala 32 17 PRTHuman immunodeficiency virus type 1 32 Thr Ser Ser Asn Thr Ala Ala ThrAsn Ala Ala Cys Ala Trp Leu Glu 1 5 10 15 Ala 33 51 DNA Humanimmunodeficiency virus type 1 33 acaagtagca atacagcagc taccaatgctgattgtgcct ggctagaagc a 51 34 17 PRT Human immunodeficiency virus type 134 Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu 1 510 15 Ala 35 51 DNA Human immunodeficiency virus type 1 CDS (1)..(42) 35aca agt agc aat aca gca gct acc aat gct gat tgt gcc tag ctagaagca 51 ThrSer Ser Asn Thr Ala Ala Thr Asn Ala Asp Cys Ala 1 5 10 36 13 PRT Humanimmunodeficiency virus type 1 36 Thr Ser Ser Asn Thr Ala Ala Thr Asn AlaAsp Cys Ala 1 5 10 37 22 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Nucleotide 37 cagaaaggca attttaggaa cc 2238 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic Nucleotide 38 cctggcttta attttactgg 20 39 17 DNA ArtificialSequence Description of Artificial Sequence Synthetic Nucleotide 39gtacagggga aagaata 17 40 25 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Nucleotide 40 ccccataata gactgtgacc cacaa25 41 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic Nucleotide 41 tgagtcgaca tgagagtgaa ggagaaatat 30 42 29 DNAArtificial Sequence Description of Artificial Sequence SyntheticNucleotide 42 tcctgcagct tatagcaaaa tcctttcca 29 43 24 DNA ArtificialSequence Description of Artificial Sequence Synthetic Nucleotide 43tcaatgccac agccatagca gtag 24 44 21 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic Nucleotide 44 cagtgggttc cctagttagc c21 45 202 DNA Human immunodeficiency virus type 1 45 atgggtggcaagtggtcaaa aagtagtgtg gttggatggc ctgctgtaag ggaaaaaatg 60 agacgagctgagccagcagc agatggggtg ggagcagtat ctcgagacct agaaaaacat 120 ggagcaatcacaagtagcaa tacagcagct accaatgctg attgtgccta gctagaagca 180 caagaggaggaggaggtggg tt 202 46 56 PRT Human immunodeficiency virus type 1 46 MetGly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Ala Val 1 5 10 15Arg Glu Lys Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30Val Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 40 45Ala Ala Thr Asn Ala Asp Cys Ala 50 55 47 202 DNA Human immunodeficiencyvirus type 1 47 atgggtggca agtggtcaaa aagtagtgtg gttggatggc ctactgtaagggaaagaatg 60 agacgagctg agccagcagc agatggggtg ggagcagcat ctcgagacctggaaaaacat 120 ggagcaatca caagtagcaa tacagcagct accaatgctg cttgtgcctggctagaagca 180 caagaggagg aggaggtggg tt 202 48 56 PRT Humanimmunodeficiency virus type 1 48 Met Gly Gly Lys Trp Ser Lys Ser Ser ValVal Gly Trp Pro Thr Val 1 5 10 15 Arg Glu Arg Met Arg Arg Ala Glu ProAla Ala Asp Gly Val Gly Ala 20 25 30 Ala Ser Arg Asp Leu Glu Lys His GlyAla Ile Thr Ser Ser Asn Thr 35 40 45 Ala Ala Thr Asn Ala Ala Cys Ala 5055

What is claimed is:
 1. A substantially pure nucleic acid molecule orfragment thereof that encodes a truncated Nef protein comprising anucleic acid sequence that encodes substantially the same sequence ofamino acid residues 1 through 56 of an HIV-1 Nef protein.
 2. The nucleicacid molecule of claim 1 wherein said nucleic acid sequence is selectedfrom the group consisting of: a nucleic acid molecule that encodessubstantially the same amino acid sequence set forth in SEQ ID NO:46;and a nucleic acid molecule that encodes substantially the same sequenceof amino acid residues 1 through 56 of an HIV-1 Nef peptide whereinamino acid residue 54 is aspartic acid.
 3. The nucleic acid molecule ofclaim 2 wherein said nucleic acid sequence is selected from the groupconsisting of: (a) nucleotides 1 through 168 of SEQ ID NO:45; and (b)nucleotides 1 through 168 of an HIV-1 Nef peptide wherein nucleotide 161is adenine.
 4. A substantially pure fragment of the nucleic acidmolecule of claim 3 comprising a nucleic acid sequence that has one ormore of the following amino acid residues of SEQ ID NO:45: guanine atnucleotide 43, adenine at nucleotide 56, thymine at nucleotide 98,adenine at nucleotide 111, and adenine at nucleotide 161 and can be usedto specifically detect the nucleic acid molecule of claim 3(a).
 5. Asubstantially pure mutated HIV-1 Nef protein fragment, comprising apeptide encoded by the nucleic acid molecule fragment of claim
 4. 6. Anantibody or fragment thereof that specifically binds to the proteinfragment of claim
 5. 7. A substantially pure truncated Nef protein orfragment thereof comprising substantially the same sequence of aminoacid residues 1 through 56 of an HIV-1 Nef protein.
 8. The truncated Nefprotein of claim 7 wherein said sequence is selected from the groupconsisting of: substantially the same amino acid sequence set forth inSEQ ID NO:46; and substantially the same sequence of amino acid residues1 through 56 of an HIV-1 Nef protein wherein amino acid residue 54 isaspartic acid.
 9. The truncated protein of claim 8 wherein said aminoacid sequence is selected from the group consisting of: amino acidresidues 1 through 56 of SEQ ID NO:46; and amino acid residues 1 through56 of an HIV-1 Nef peptide wherein amino acid residue 54 is asparticacid.
 10. An antibody or fragment thereof that specifically binds to themutated protein of claim 7, 8 or
 9. 11. A substantially pure nucleicacid molecule or fragment thereof that encodes a mutated HIV-1 Env gp41protein containing an arginine at amino acid residue
 660. 12. Thenucleic acid molecule of claim 11 wherein nucleotide 1979 is guanine.13. A substantially pure fragment of the nucleic acid molecule of claim12 comprising a portion of said nucleic acid molecule that contains saidguanine at nucleotide 1979 and can be used to specifically detect thenucleic acid molecule of claim
 12. 14. A substantially pure mutatedHIV-1 Env gp41 protein fragment, comprising a peptide encoded by thenucleic acid molecule fragment of claim
 13. 15. An antibody or fragmentthereof that specifically binds to the protein fragment of claim
 14. 16.A substantially pure mutated HIV-1 Env gp41 protein or fragment thereofcomprising an HIV-1 Env gp41 protein containing an arginine at aminoacid residue
 660. 17. An antibody or fragment thereof that specificallybinds to the protein of claim
 16. 18. A substantially pure nucleic acidmolecule or fragment thereof that encodes a mutated HIV-1 Env gp120protein selected from the group consisting of: (a) a nucleic acidsequence that encodes an HIV-1 Env gp120 protein wherein: amino acidresidue 143 is either serine or arginine; up to eight amino acids atamino acid residues 144 through 151 in the V1 domain are deleted; andamino acid residue 153 is either methionine or isoleucine; and (b) anucleic acid sequence that encodes an HIV-1 Env gp120 protein wherein:amino acid residue 187 is either isoleucine or valine; and one or twoamino acids at amino acid residues 192 and 193 in the V2 domain aredeleted.
 19. A substantially pure fragment of the nucleic acid moleculeof claim 18 comprising a portion of the nucleic acid molecule thatcontains said deletions and that can be used to specifically detect thenucleic acid molecule of claim
 18. 20. A substantially pure mutatedHIV-1 Env gp120 protein fragment, comprising a peptide encoded by thenucleic acid molecule fragment of claim
 19. 21. An antibody or fragmentthereof that specifically binds to the protein fragment of claim
 20. 22.A substantially pure mutated HIV-1 Env gp120 protein or fragment thereofselected from the group consisting of: (a) the amino acid sequence of anHIV-1 Env gp120 protein wherein amino acid residue 143 is either serineor arginine; up to eight amino acids at amino acid residues 144 through151 in the V1 domain are deleted; and amino acid residue 153 is eithermethionine or isoleucine; and (b) the amino acid sequence of an HIV-1Env gp120 protein wherein amino acid residue 187 is either isoleucine orvaline; and one or two amino acids at amino acid residues 192 and 193 inthe V2 domain are deleted.
 23. An antibody or fragment thereof thatspecifically binds to the protein of claim
 22. 24. A substantially purenucleic acid molecule or fragment thereof that encodes a truncated HIV-1Vpr protein comprising a nucleic acid sequence encoding substantiallythe same amino acid residues 1 through 17 of an HIV-1 Vpr protein. 25.The nucleic acid molecule of claim 24 wherein said nucleic acid sequencecomprises nucleotides 1 through 51 of an HIV-1 vpr gene.
 26. Asubstantially pure truncated HIV-1 Vpr protein or fragment thereofcomprising substantially the same amino acid residues 1 through 17 of anHIV-1 Vpr protein.
 27. An antibody or fragment thereof that specificallybinds to the protein of claim
 26. 28. A substantially pure nucleic acidmolecule or fragment thereof that encodes a mutated and truncated HIV-1Pol protease protein comprising a nucleic acid sequence encodingsubstantially the same sequence of amino acid residues 1 through 13 of awild-type HIV-1 Pol protease protein and amino acid residues 14 though29 of SEQ ID NO:6.
 29. The nucleic acid molecule of claim 26 whereinsaid nucleic acid sequence comprises nucleotides 1 through 41 of awild-type HIV-1 Pol protease gene and nucleotides 42 though 87 of SEQ IDNO:5.
 30. A substantially pure fragment of the nucleic acid molecule ofclaim 29, comprising a fragment of SEQ ID NO:5 at least 8 nucleotides inlength and that can be used to specifically detect the nucleic acidmolecule of claim
 29. 31. A substantially pure mutated and truncatedHIV-1 Pol protease protein or fragment thereof comprising substantiallythe same sequence of amino acid residues 1 through 13 of a wild-typeHIV-1 Pol protease protein and amino acid residues 14 though 29 of SEQID NO:6.
 32. An immunogen comprising an inactivated protease-defectiveviral HIV-1 particle containing one or more of the followingsubstantially pure proteins: an HIV-1 Env gp120 protein comprisingsubstantially the same protein of claim 22; a mutated HIV-1 Pol proteasemolecule comprising substantially the same sequence of amino acidresidues 1 through 13 of a wild-type HIV-1 Pol protease protein andamino acid residues 14 though 29 of SEQ ID NO:6; a truncated HIV-1 Nefprotein or fragment thereof comprising substantially the same sequenceof amino acid residues 1 though 56 of an HIV-1 Nef protein; a mutatedHIV-1 Nef protein comprising substantially the same amino acid sequenceset forth in SEQ ID NO:46 or a fragment thereof comprising one or moreof the following amino acid residues of SEQ ID NO:46: alanine at aminoacid residue 15, lysine at amino acid residue 19, valine at amino acidresidue 33, and aspartic acid at amino acid residue 54; a truncatedHIV-1 Nef protein comprising substantially the same sequence of aminoacid residues 1 through 56 of a Nef peptide wherein amino acid residue54 is aspartic acid; a truncated HIV-1 Vpr protein or fragment thereofcomprising substantially the same amino acid residues 1 through 17 of anHIV-1 Vpr protein; or an HIV-1 Env gp41 protein or fragment thereofcomprising substantially the same amino acid sequence of an HIV-1 Envgp41 protein containing an arginine at amino acid residue
 660. 33. Theimmunogen of claim 32 further comprising one or more suitable adjuvants.34. A method of detecting a mutated HIV-1 gene selected from the groupconsisting of a nef gene, an env gp41 gene and an env gp120 gene orfragments thereof in a sample of cells, lysed cells or extracellularfluid from an individual, comprising the steps of: obtaining from anindividual a sample of cells, lysed cells or extracellular fluidsuspected of containing said mutated gene or fragments thereof;contacting said sample with a measured amount of the nucleic acidmolecule of claims 4, 13 or 19, respectively, to hybridize with saidgene or fragment thereof; and determining the presence of saidhybridized molecule to detect the presence of the mutated gene orfragments thereof in said sample.
 35. A method of detecting the presenceof a mutated HIV-1 protein selected from the group consisting of a Nefprotein, an Env gp41 protein and an Env gp120 protein or fragmentsthereof in an individual, comprising the steps of: obtaining from anindividual a sample of cells, lysed cells or extracellular fluidsuspected of containing a mutated HIV-1 protein or fragments thereof;contacting said sample with a measured amount of the antibody of claims6 or 10, 15 or 17, or 21 or 23, respectively, to form a complex of theantibody and said protein or fragment thereof; and determining thepresence of said complex to detect the presence of the mutated proteinor fragments thereof in said sample.
 36. A method of reducing theseverity of HIV-1 infections in individuals, comprising contactingprotease-defective viral particles containing one or more mutated HIV-1Nef proteins, mutated HIV-1 Env gp41 proteins, or mutated HIV-1 Envgp120 proteins or fragments thereof present on cells, in lysed cells orin extracellular fluid with an effective amount of the antibody ofclaims 6 or 10, 15 or 17, or 21 or 23, respectively, to inhibit theactivity of said proteins or fragments thereof.
 37. The method of claim36 wherein said antibody is attached to a moiety selected from the groupconsisting of radioactive moieties, chemotherapeutic moieties andchemotoxic moieties.
 38. A method of reducing or preventing apoptoticcell lysis in an HIV-1 seropositive or seronegative individual,comprising administering an immunologically effective amount of theimmunogen of claim 32 or 33 to the individual.